当归补血汤对血管性痴呆模型大鼠PINK1/Parkin信号通路的影响OA

Objective:To investigate the potential mechanism of Danggui Buxuetang(DBT)in the treatment of vascular dementia(VAD).Methods:Sixty male SD rats were randomly assigned to the sham-operated group,model group,DBT low-,medium-,and high-dose groups,and the donepezil group.Except for the sham-operated group,rats in all other groups underwent bilateral common carotid artery ligation.After successful modeling,DBT was administered at doses of 9.2,18.4,36.8 g·kg-1 for the low-,medium-,and high-dose groups,respectively,while the donepezil group received 3 mg·kg-1 donepezil solution by gavage once daily.After 4 consecutive weeks of drug treatment,rats underwent the Morris water maze test,novel object recognition test,Nissl staining to observe hippocampal neurons,and immunofluorescence staining to detect the expression of neuronal nuclear protein(NeuN)in the hippocampus.Western blot was used to assess the expression of PTEN-induced kinase 1(PINK1),Parkin,microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ),B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax).Transmission electron microscopy was used to observe hippocampal neuronal ultrastructure.Real-time PCR was used to detect the expression of NADPH oxidase subunits p22phox and p47phox in hippocampal tissues.The levels of malondialdehyde(MDA),glutathione(GSH),superoxide dismutase(SOD),and total antioxidant capacity were measured to evaluate oxidative stress levels.Results:In the Morris water maze test,escape latency changed significantly over time in all groups except the model group.Compared with the sham-operated group,the model group showed significantly prolonged escape latency(P<0.01).Compared with the model group,rats in the DBT groups and the donepezil group exhibited significantly shorter escape latency(P<0.05,P<0.01).The number of crossings over the original platform was significantly reduced in the model group compared with the sham-operated group(P<0.01),whereas rats in the DBT and donepezil groups showed significantly increased platform crossings compared with the model group(P<0.05,P<0.01).Compared with the sham-operated group,exploration time of new objects was significantly reduced in the model group(P<0.01).Compared with the model group,exploration time of new objects increased significantly in the medium-and high-dose DBT groups and the donepezil group(P<0.05,P<0.01),while no significant change was observed in the low-dose DBT group.Compared with the high-dose DBT group,rats in the donepezil group had significantly prolonged escape latency and reduced platform crossings and new-object exploration time(P<0.05).Nissl staining showed decreased density of healthy neurons in the CA1 and CA3 regions of the hippocampus in the model group,with loss of Nissl bodies and nuclear atrophy or disappearance.In the high-dose DBT group,neuronal density in CA1 and CA3 increased,with neurons arranged closely and displaying normal morphology.Immunofluorescence showed that compared with the sham-operated group,the hippocampal NeuN⁺ cell count in the VAD model group was significantly decreased(P<0.01),compared with the VAD model group,the hippocampal NeuN⁺ cell count in the high-dose DBT group was significantly increased(P<0.01).Compared with the sham-operated group,the expression of PINK1,Parkin,LC3Ⅱ,and Bax proteins was significantly increased(P<0.01),while the expression of Bcl-2 was significantly decreased in the VAD model group(P<0.01).Compared with the VAD model group,the high-dose DBT group showed significantly decreased expression of PINK1,Parkin,LC3Ⅱ,and Bax proteins(P<0.01)and significantly upregulated Bcl-2 expression(P<0.01).The medium-dose DBT group exhibited significantly reduced expression of Parkin,LC3Ⅱ,and Bax proteins(P<0.05,P<0.01)and significantly increased Bcl-2 expression(P<0.01),while no statistically significant differences were observed in the low-dose DBT group.Transmission electron microscopy showed mitochondrial pyknosis,thickened cristae,increased electron density,and the presence of mitochondrial autophagy in the model group.In contrast,hippocampal neurons in the high-dose DBT group contained abundant mitochondria with intact morphology,clear cristae,and uniform matrix.Compared with the sham-operated group,total antioxidant capacity,SOD activity,and GSH levels were significantly decreased,while MDA levels were significantly increased in the model group(P<0.01).Compared with the model group,total antioxidant capacity and antioxidant levels(SOD,GSH)increased significantly,and MDA decreased significantly in the medium-and high-dose DBT groups(P<0.01),while no significant changes were observed in the low-dose DBT group.Compared with the sham-operated group,mRNA expression of p22phox and p47phox was significantly increased in the model group(P<0.01).Compared with the model group,expression of p22phox and p47phox was significantly decreased in the DBT groups(P<0.05,P<0.01).Conclusion:DBT may exert neuroprotective effects by regulating PINK1/Parkin-mediated mitochondrial autophagy,thereby improving learning and memory abilities and treating VAD.